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PubMed2004

Lack of phylogeography in European mammals before the last glaciation.

In many extant animal and plant species in Europe and North America a correlation exists between the geographical location of individuals and the genetic relatedness of the mitochondrial (mt) DNA sequences that they carry. Here, we analyze mtDNA sequences from cave bears, brown bears, cave hyenas, and Neandertals in Europe before the last glacial maximum and fail to detect any phylogeographic patterns similar to those observed in extant species. We suggest that at the beginning of the last glacial maximum, little phylogeographic patterns existed in European mammals over most of their geographical ranges and that current phylogeographic patterns are transient relics of the last glaciation. Cycles of retreat of species in refugia during glacial periods followed by incomplete dispersal from one refugium into other refugia during interglacial periods is likely to be responsible for the deep genetic divergences between phylogeographic clusters of mtDNA seen today.

Animals↗

PubMed2006

Evolution of rattlesnakes (Viperidae; Crotalus) in the warm deserts of western North America shaped by Neogene vicariance and Quaternary climate change.

During Pleistocene, the Laurentide ice sheet rearranged and diversified biotic distributions in eastern North America, yet had minimal physical impact in western North America where lineage diversification is instead hypothesized to result from climatic changes. If Pleistocene climatic fluctuations impacted desert species, the latter would reflect patterns of restricted gene flow concomitant with indications of demographic bottlenecks. Accordingly, molecular evidence for refugia should be present within these distributions and for subsequent range expansions as conditions improved. We sought answers to these questions by evaluating mitochondrial DNA (mtDNA) sequences from four species of rattlesnakes [Crotalus mitchellii (speckled rattlesnake), Crotalus cerastes (sidewinder), Crotalus tigris (tiger rattlesnake), Crotalus ruber (red diamond rattlesnake)] with distributions restricted to desert regions of southwestern North America. We inferred relationships using parsimony and maximum likelihood, tested intraspecific clades for population expansions, applied an isolation-with-migration model to determine bi-directional migration rates (m) among regions, and inferred divergence times for species and clades by applying a semiparametric penalized likelihood approach to our molecular data. Evidence for significant range expansion was present in two of eight regions in two species (Crotalus mitchellii pyrrhus, C. tigris region north). Two species (C. cerastes, C. mitchellii) showed a distribution concomitant with northward displacement of Baja California from mainland México, followed by vicariant separation into subclades. Effects of Pleistocene climate fluctuations were found in the distributions of all four species. Three regional diversification patterns were identified: (i) shallow genetic diversity that resulted from Pleistocene climatic events (C. tigris, C. ruber); (ii) deep Pleistocene divisions indicating allopatric segregation of subclades within refugia (C. mitchellii, C. cerastes); and (iii) lineage diversifications that extended to Pliocene or Late Miocene (C. mitchellii, C. cerastes). Clade-diversifying and clade-constraining effects impacted the four species of rattlesnakes unequally. We found relatively high levels of molecular diversification in the two most broadly distributed species (C. mitchellii, C. cerastes), and lower levels of genetic diversification in the two species (C. tigris, C. ruber) whose ranges are relatively more restricted. Furthermore, in several cases, the distributions of subspecies were not congruent with our molecular information. We suggest regional conservation perspectives for southwestern deserts cannot rely upon subspecies as biodiversity surrogates, but must instead employ a molecular and deep historical perspective as a primary mechanism to frame biodiversity reserves within this region.

Adenosine Triphosphatases↗

PubMed1991

Activation of methionine by Escherichia coli methionyl-tRNA synthetase.

In the present work, we have examined the function of three amino acid residues in the active site of Escherichia coli methionyl-tRNA synthetase (MetRS) in substrate binding and catalysis using site-directed mutagenesis. Conversion of Asp52 to Ala resulted in a 10,000-fold decrease in the rate of ATP-PPi exchange catalyzed by MetRS with little or no effect on the Km's for methionine or ATP or on the Km for the cognate tRNA in the aminoacylation reaction. Substitution of the side chain of Arg233 with that of Gln resulted in a 25-fold increase in the Km for methionine and a 2000-fold decrease in kcat for ATP-PPi exchange, with no change in the Km for ATP or tRNA. These results indicate that Asp52 and Arg233 play important roles in stabilization of the transition state for methionyl adenylate formation, possibly directly interacting with complementary charged groups (ammonium and carboxyl) on the bound amino acid. Primary sequence comparisons of class I aminoacyl-tRNA synthetases show that all but one member of this group of enzymes has an aspartic acid residue at the site corresponding to Asp52 in MetRS. The synthetases most closely related to MetRS (including those specific for Ile, Leu, and Val) also have a conserved arginine residue at the position corresponding to Arg233, suggesting that these conserved amino acids may play analogous roles in the activation reaction catalyzed by each of these enzymes. Trp305 is located in a pocket deep within the active site of MetRS that has been postulated to form the binding cleft for the methionine side chain.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Monophosphate↗

PubMed2024

Whole-genome sequencing in 333,100 individuals reveals rare non-coding single variant and aggregate associations with height.

The role of rare non-coding variation in complex human phenotypes is still largely unknown. To elucidate the impact of rare variants in regulatory elements, we performed a whole-genome sequencing association analysis for height using 333,100 individuals from three datasets: UK Biobank (N = 200,003), TOPMed (N = 87,652) and All of Us (N = 45,445). We performed rare ( < 0.1% minor-allele-frequency) single-variant and aggregate testing of non-coding variants in regulatory regions based on proximal-regulatory, intergenic-regulatory and deep-intronic annotation. We observed 29 independent variants associated with height at P < after conditioning on previously reported variants, with effect sizes ranging from -7cm to +4.7 cm. We also identified and replicated non-coding aggregate-based associations proximal to HMGA1 containing variants associated with a 5 cm taller height and of highly-conserved variants in MIR497HG on chromosome 17. We have developed an approach for identifying non-coding rare variants in regulatory regions with large effects from whole-genome sequencing data associated with complex traits.

Humans↗

PubMed1994

Structure and distribution of N-cadherin in developing zebrafish embryos: morphogenetic effects of ectopic over-expression.

N-cadherin cDNA was cloned from a zebrafish embryonic cDNA library. Analysis of the deduced amino acid sequence of this molecule (ZN-cadherin) revealed a high degree of homology to N-cadherins of other species, except that its pre-sequence is considerably shorter. Nevertheless, following transfection into chinese hamster ovary (CHO) cells, the expressed protein was functionally active, namely participated in calcium-dependent intercellular interactions. Moreover, ectopic over-expression of ZN-cadherin, following mRNA microinjection into 2-4 cell embryos, caused microaggregation and uneven segregation of deep cells, resulting in distorted embryos. Developmental Northern and Western blot analyses indicated that both the mRNA and the protein first appear at gastrulation. In-situ hybridization showed that ZN-cadherin mRNA was initially present in all deep cells, and later became restricted to various epithelial and neural tissues. Whole-mount immunostaining indicated that while ZN-cadherin was already present at 50% epiboly, it became associated with cell junctions only 4-5 h later. In developing somites ZN-cadherin expression was prominent but transient. High levels of the protein were detected in epithelial somites and its expression was apparently down regulated concomitantly with the onset of myogenesis.

Amino Acid Sequence↗

PubMed2006

Bathymetric and geographic population structure in the pan-Atlantic deep-sea bivalve Deminucula atacellana (Schenck, 1939).

The deep-sea soft-sediment environment hosts a diverse and highly endemic fauna of uncertain origin. We know little about how this fauna evolved because geographic patterns of genetic variation, the essential information for inferring patterns of population differentiation and speciation are poorly understood. Using formalin-fixed specimens from archival collections, we quantify patterns of genetic variation in the protobranch bivalve Deminucula atacellana, a species widespread throughout the Atlantic Ocean at bathyal and abyssal depths. Samples were taken from 18 localities in the North American, West European and Argentine basins. A hypervariable region of mitochondrial 16S rDNA was amplified by polymerase chain reaction (PCR) and sequenced from 130 individuals revealing 21 haplotypes. Except for several important exceptions, haplotypes are unique to each basin. Overall gene diversity is high (h = 0.73) with pronounced population structure (Phi(ST) = 0.877) and highly significant geographic associations (P < 0.0001). Sequences cluster into four major clades corresponding to differences in geography and depth. Genetic divergence was much greater among populations at different depths within the same basin, than among those at similar depths but separated by thousands of kilometres. Isolation by distance probably explains much of the interbasin variation. Depth-related divergence may reflect historical patterns of colonization or strong environmental selective gradients. Broadly distributed deep-sea organisms can possess highly genetically divergent populations, despite the lack of any morphological divergence.

Animals↗

PubMed2001

Deep molecular divergence in the absence of morphological and ecological change in the Californian coastal dune endemic trapdoor spider Aptostichus simus.

Aptostichus simus is a trapdoor spider endemic to the coastal dunes of central and southern California and, on morphological grounds, is recognized as a single species. Mitochondrial DNA 16S rRNA sequences demonstrate that most populations are fixed for the same haplotype and that the population haplotypes from San Diego County, Los Angeles County, Santa Rosa Island, and Monterey County are extremely divergent (6-12%), with estimated separation times ranging from 2 to 6 million years. A statistical cluster analysis of morphological features demonstrates that this genetic divergence is not reflected in anatomical features that might signify ecological differentiation among these lineages. The species status of these divergent populations of A. simus depends upon the species concept utilized. If a time-limited genealogical perspective is employed, A. simus would be separated at the base into two genetically distinct species. This study suggests that species concepts based on morphological distinctiveness, in spider groups with limited dispersal capabilities, probably underestimate true evolutionary diversity.

Animals↗

PubMed1995

Analysis and clinical implications of K-ras gene mutations and infection with human papillomavirus types 16 and 18 in primary adenocarcinoma of the uterine cervix.

Experimental models indicate that activated ras genes and HPV oncogenic sequences may cooperate in inducing a completely transformed phenotype in epithelial cells. We searched for K-ras gene mutations and HPV type-16 and -18 sequences in 67 primary adenocarcinomas of the uterine cervix by analyzing DNAs from formalin-fixed, paraffin-embedded tissue samples. Target sequences were amplified by PCR and analyzed by denaturing gradient gel electrophoresis (DGGE) and sequencing for the detection of K-ras gene mutations and by Southern blotting for the detection of HPV infection. We found 16 mutations in 15 cases; 14 were at codon 12 and 2 at codon 13; 11 were base transitions and 5 were transversions. Mutations were more frequent in mucin-secreting than in non-mucinous tumors. HPV oncogenic sequences were detected in 58 cases with no significant difference between K-ras-mutated and wild-type tumors. HPV oncogenic sequences were also more frequent in mucin-secreting than in non-mucinous tumors. Both molecular events were present simultaneously in 13 out of 58 cases, all of which had histologically grade-2 and grade-3 tumors. Clinico-pathological parameters of the disease and the overall survival, however, were independent of K-ras mutations and of HPV-16 and -18 infection, as shown by univariate and multivariate analysis. In contrast, stage of disease, lymph-node metastases, deep infiltration, clear-cell histology and low grade of differentiation were risk factors for tumor-related death.

Adenocarcinoma↗

PubMed1997

Phylogenetic relationships of the liverworts (Hepaticae), a basal embryophyte lineage, inferred from nucleotide sequence data of the chloroplast gene rbcL.

Sequence data from the chloroplast-encoded gene rbcL were obtained for 24 liverworts, a basal group of embryophytes. Maximum likelihood and parsimony analyses of these data, along with data from other major green plant lineages, confirm hypotheses based on morphological data, such as the paraphyly of bryophytes, and the basal position of liverworts. Molecular data corroborate the deep separation between the complex thalloid and leafy/simple thalloid liverworts implied by morphological data, but the monophyly of liverworts could not be rejected. The effects of accounting for site-to-site rate heterogeneity in these data were examined using maximum likelihood methods. Comparison of trees obtained with and without rate heterogeneity showed that simply allowing for heterogeneity had a greater improvement on likelihood score than optimization of transition/transversion bias. Incorporation of site-to-site rate heterogeneity in the larger analysis, however, did not necessarily change which topology was favored. Properties of rbcL sequences from the two liverwort groups were compared. Significantly different substitution rates were found between leafy/simple thalloid and complex thalloid liverwort taxa, with rates of rbcL sequence evolution in leafy/simple thalloid taxa being higher and more indicative of those of vascular plants, and with those of complex thalloid taxa (such as Marchantia) being slower. Codon usage in rbcL in complex thalloid liverworts was biased toward NNU and NNA, compared to the leafy/simple thalloid liverworts. Although base composition and relative substitution rates differed between the two groups, no significant differences were detected within each of the two groups of liverworts. The signal present in first and second codon sites versus third codon sites was compared. While the third codon positions in rbcL across this taxon sampling are highly variable (with only 15 constant sites of 439), the trees obtained were in general agreement with trees from the entire data set and with trees obtained from independent sources of data. The presence of signal in third codon positions across greater than 400 MY of plant evolution means that definitions of saturation based on pair-wise comparisons of sequences inadequately assess phylogenetic signal.

Chloroplasts↗

PubMed1995

Pattern of muscle fiber type formation in the pig.

The aim of this study was to analyze the temporal sequence of expression of the myosin isoforms in the populations of muscle fibers in the pig and to bring more information on the origin of the strikingly different pattern of fiber composition and distribution between the deep medial red (oxido-glycolytic) and superficial white (glycolytic) portions of semitendinosus (ST) muscle. Muscle samples were taken from 49-, 55-, 75-, 90-, 103-, and 113- (birth) day-old fetuses, from 6-, 11-, 21-, 35-, 50-, and 80-day-old piglets, and from a 3-year-old pig. Our results confirm the sequential formation of primary and secondary generation fibers. The use of immunohistochemistry and heterologous monoclonal antibodies (mAb) directed against specific myosin heavy chain (MHC) isoforms revealed a different pattern of gene expression between the two portions of the ST muscle for both generations of fibers. By 75 days of gestation (dg), primary myotubes from the deep medial portion stained positively for the anti-slow MHC mAb and negatively for the adult anti-fast MHC, whereas the opposite was observed in the superficial portion. Secondary fibers never expressed slow MHC until late gestation. Instead, they expressed an adult fast MHC isoform as soon as they formed in the deep medial portion and later on in the superficial portion. From late gestation to the first 3 postnatal weeks, slow MHC began to be expressed in a subpopulation of secondary fibers. These fibers were in the direct vicinity of primary myotubes in the deep medial portion, whereas their location could not be established in the superficial portion. The remaining secondary fibers matured to type IIA in the direct vicinity of these type I fibers and to type IIB at the periphery of the islets. In both portions of the muscle, a subpopulation of secondary fibers, the first ones to express slow MHC, also transitorily expressed a MHC that was identical or closely related to the alpha-cardiac MHC during the early postnatal period. A third generation of small diameter fibers was observed shortly after birth and reacted with the anti-fetal MHC mAb; their destiny remains to be established.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1993

An electrostatic mechanism for substrate guidance down the aromatic gorge of acetylcholinesterase.

Electrostatic calculations based on the recently solved crystal structure of acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) indicate that this enzyme has a strong electrostatic dipole. The dipole is aligned with the gorge leading to its active site, so that a positively charged substrate will be drawn to the active site by its electrostatic field. Within the gorge, aromatic side chains appear to shield the substrate from direct interaction with most of the negatively charged residues that give rise to the dipole. The affinity of quaternary ammonium compounds for aromatic rings, coupled with this electrostatic force, may work in concert to create a selective and efficient substrate-binding site in acetylcholinesterase and explain why the active site is situated at the bottom of a deep gorge lined with aromatic residues.

Acetylcholinesterase↗

PubMed1986

Optimisation of indices of external pneumatic compression for prophylaxis against deep vein thrombosis: radionuclide gated imaging studies.

The amount and rate of blood expelled with different modes of intermittent external pneumatic compression applied to the lower leg were studied on a regional basis in a series of experiments on healthy human volunteers. Radionuclide imaging of the labelled blood pool, with acquisition of counts synchronised to the pressurisation cycle, provided data on regional blood volumes in the leg in relation to time. To determine the changes in blood volume of the lower leg resulting from external pneumatic compression labelled red blood cell counts were determined during 10 different types of compression cycle. Since venous stasis is considered to be a major cause of venous thrombosis the red blood cell counts were used to calculate regional values of the fraction of blood ejected as well as comparative indices proportional to regional flow rate, regional velocity, and regional wall shear stress. All these indices should be maximised for optimal prophylaxis against deep vein thrombosis. The four compartment cuff in each compression mode applied a mean pressure of 45 mm Hg, but different combinations of values were used for intercompartmental pressure gradation (delta p) and for intercompartmental time sequencing to the onset of compression (delta t). Uniform compression (delta p = 0; delta t = 0) was substantially inferior to cycles with gradation and sequencing. The optimal values of delta p were in the range 5-10 mm Hg and of delta t in the range 0-0.5 seconds.

Adolescent↗

PubMed2004

Covarion shifts cause a long-branch attraction artifact that unites microsporidia and archaebacteria in EF-1alpha phylogenies.

Microsporidia branch at the base of eukaryotic phylogenies inferred from translation elongation factor 1alpha (EF-1alpha) sequences. Because these parasitic eukaryotes are fungi (or close relatives of fungi), it is widely accepted that fast-evolving microsporidian sequences are artifactually "attracted" to the long branch leading to the archaebacterial (outgroup) sequences ("long-branch attraction," or "LBA"). However, no previous studies have explicitly determined the reason(s) why the artifactual allegiance of microsporidia and archaebacteria ("M + A") is recovered by all phylogenetic methods, including maximum likelihood, a method that is supposed to be resistant to classical LBA. Here we show that the M + A affinity can be attributed to those alignment sites associated with large differences in evolutionary site rates between the eukaryotic and archaebacterial subtrees. Therefore, failure to model the significant evolutionary rate distribution differences (covarion shifts) between the ingroup and outgroup sequences is apparently responsible for the artifactual basal position of microsporidia in phylogenetic analyses of EF-1alpha sequences. Currently, no evolutionary model that accounts for discrete changes in the site rate distribution on particular branches is available for either protein or nucleotide level phylogenetic analysis, so the same artifacts may affect many other "deep" phylogenies. Furthermore, given the relative similarity of the site rate patterns of microsporidian and archaebacterial EF-1alpha proteins ("parallel site rate variation"), we suggest that the microsporidian orthologs may have lost some eukaryotic EF-1alpha-specific nontranslational functions, exemplifying the extreme degree of reduction in this parasitic lineage.

Animals↗

PubMedDate not supplied

Osteoarthritic changes at the severely degenerative disc in humans.

A series of postmortem spine specimens demonstrated new osteophytic cartilage production at levels of severe disc degeneration. This tissue is related to the cartilage found within the intervertebral disc. The sequence of pathologic changes occurring within these discs is shown radiographically and histopathologically. A cleft occurred between adjacent vertebral bodies, and recognizable disc material was absent at the cleft. The adjacent bone showed sclerosis, even extending deep within the bodies. Cartilage changes near the two end-plates resemble the histology of degenerative arthritis of hyaline cartilage of a synovial joint.

Aged↗

PubMed2000

The 1.9 A crystal structure of Escherichia coli MurG, a membrane-associated glycosyltransferase involved in peptidoglycan biosynthesis.

The 1.9 A X-ray structure of a membrane-associated glycosyltransferase involved in peptidoglycan biosynthesis is reported. This enzyme, MurG, contains two alpha/beta open sheet domains separated by a deep cleft. Structural analysis suggests that the C-terminal domain contains the UDP-GlcNAc binding site while the N-terminal domain contains the acceptor binding site and likely membrane association site. Combined with sequence data from other MurG homologs, this structure provides insight into the residues that are important in substrate binding and catalysis. We have also noted that a conserved region found in many UDP-sugar transferases maps to a beta/alpha/beta/alpha supersecondary structural motif in the donor binding region of MurG, an observation that may be helpful in glycosyltransferase structure prediction. The identification of a conserved structural motif involved in donor binding in different UDP-sugar transferases also suggests that it may be possible to identify--and perhaps alter--the residues that help determine donor specificity.

Amino Acid Sequence↗

PubMed2006

Hybridization between mtDNA-defined phylogeographic lineages of black ratsnakes (Pantherophis sp.).

Phylogeographic analyses using mitochondrial DNA (mtDNA) have revealed many examples of apparently deep historical subdivisions ('phylogroups') within many vertebrates. It remains unclear whether these phylogroups represent independently evolving, adaptively differentiated lineages or groups that show little functional differentiation and, hence, will merge on contact. Here, we use mtDNA sequence data to evaluate the phylogeographic relationships between two of the northernmost populations of black ratsnakes (Pantherophis obsoletus complex) in Ontario, Canada and previously analysed populations in the United States. We then use population-level analyses to evaluate the level of adaptive divergence between previously established mtDNA phylogroups. Phylogenetic analyses show that southern Ontario snakes have mtDNA haplotypes that fall within the Central mtDNA phylogroup, as designated by Burbrink et al. (2000). In contrast, snakes in eastern Ontario carry either Central or Eastern-specific haplotypes. Within the hybrid region, we found highly variable frequencies of mtDNA haplotypes among isolated sub-populations, no association between variation in cytonuclear (mtDNA) and nuclear (microsatellite DNA) markers, no difference in survival or reproductive success among snakes with different mtDNA haplotypes, and no effect of mate similarity in mtDNA on female clutch size. These results argue that the Eastern and Central phylogroups have merged in this region, likely due to a lack of adaptive differentiation between individuals in each lineage. Hence, in these snakes, phylogeographic structure in mtDNA is more a reflection of historical isolation rather than adaptive divergence. The observed reticulation between lineages and lack of evidence for hybrid disgenesis also bears on the classification of these lineages as distinct species.

Adaptation, Physiological↗

PubMed2004

Spatial distribution of marine crenarchaeota group I in the vicinity of deep-sea hydrothermal systems.

Distribution profiles of marine crenarchaeota group I in the vicinity of deep-sea hydrothermal systems were mapped with culture-independent molecular techniques. Planktonic samples were obtained from the waters surrounding two geographically and geologically distinct hydrothermal systems, and the abundance of marine crenarchaeota group I was examined by 16S ribosomal DNA clone analysis, quantitative PCR, and whole-cell fluorescence in situ hybridization. A much higher proportion of marine crenarchaeota group I within the microbial community was detected in deep-sea hydrothermal environments than in normal deep and surface seawaters. The highest proportion was always obtained from the ambient seawater adjacent to hydrothermal emissions and chimneys but not from the hydrothermal plumes. These profiles were markedly different from the profiles of epsilon-Proteobacteria, which are abundant in the low temperatures of deep-sea hydrothermal environments.

Base Sequence↗

PubMed2004

Novel forms of structural integration between microbes and a hydrothermal vent gastropod from the Indian Ocean.

Here we describe novel forms of structural integration between endo- and episymbiotic microbes and an unusual new species of snail from hydrothermal vents in the Indian Ocean. The snail houses a dense population of gamma-proteobacteria within the cells of its greatly enlarged esophageal gland. This tissue setting differs from that of all other vent mollusks, which harbor sulfur-oxidizing endosymbionts in their gills. The significantly reduced digestive tract, the isotopic signatures of the snail tissues, and the presence of internal bacteria suggest a dependence on chemoautotrophy for nutrition. Most notably, this snail is unique in having a dense coat of mineralized scales covering the sides of its foot, a feature seen in no other living metazoan. The scales are coated with iron sulfides (pyrite and greigite) and heavily colonized by epsilon- and delta-proteobacteria, likely participating in mineralization of the sclerites. This novel metazoan-microbial collaboration illustrates the great potential of organismal adaptation in chemically and physically challenging deep-sea environments.

Animals↗